Project description:Aimed to facilitate candidate biomarkers selection and improve network-based multi-target therapy, we perform comparative proteomics research on muscle-invasive bladder transitional cell carcinoma. Laser capture microdissection was used to harvest purified muscle-invasive bladder cancer cells and normal urothelial cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature.A total of 885/890 proteins commonly appeared in 4 paired samples. 295/337 of the 488/493 proteins that specific expressed in tumor/normal cells own gene ontology (GO) cellular component annotation. Compared with the entire list of the international protein index (IPI), there are 42/45 GO terms exhibited as enriched and 9/5 exhibited as depleted, respectively. Several pathways exhibit significantly changes between cancer and normal cells, mainly including spliceosome, endocytosis, oxidative phosphorylation, etc. Finally, descriptive statistics show that the PI Distribution of candidate biomarkers have certain regularity.The present study identified the proteome expression profile of muscle-invasive bladder cancer cells and normal urothelial cells, providing information for subcellular pattern research of cancer and offer candidate proteins for biomarker panel and network-based multi-target therapy.
Project description:The H3K27me3 ChIP-seq data for the human bladder transitional cell carcinoma cell line CL1207 were generated in order to detect regions of regional epigenetic silencing in this cell line and test the performance of several peak calling tools: CCAT (Xu et al., 2010) and HMCan (Ashoor et al., "HMCan – a tool to detect chromatin modifications in cancer samples using ChIP-seq data", submitted). Overall design: The human bladder cancer cell line CL1207 was derived from a muscle-invasive bladder cancer (De Boer et al., 1997). 5x105 cells were immunoprecipitated per ChIP assay with 4 μg of rabbit polyclonal antibodies against trimethyl histone H3 lysine 27 (Upstate Biotechnology, Santa Cruz, CA) and Dynabeads® Protein A (Invitrogen, Cergy Pontoise, France) in dilution buffer containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl at pH 8.0, and protease inhibitors. Six ChIP assays in the same experimental conditions were necessary to perform one ChIP-Seq experiment, so the total of 3x106 cells for each of the duplicates.
Project description:The H3K27me3 ChIP-seq data for the human bladder transitional cell carcinoma cell line CL1207 were generated in order to detect regions of regional epigenetic silencing in this cell line and test the performance of several peak calling tools: CCAT (Xu et al., 2010) and HMCan (Ashoor et al., "HMCan – a tool to detect chromatin modifications in cancer samples using ChIP-seq data", submitted). The human bladder cancer cell line CL1207 was derived from a muscle-invasive bladder cancer (De Boer et al., 1997). 5x105 cells were immunoprecipitated per ChIP assay with 4 μg of rabbit polyclonal antibodies against trimethyl histone H3 lysine 27 (Upstate Biotechnology, Santa Cruz, CA) and Dynabeads® Protein A (Invitrogen, Cergy Pontoise, France) in dilution buffer containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl at pH 8.0, and protease inhibitors. Six ChIP assays in the same experimental conditions were necessary to perform one ChIP-Seq experiment, so the total of 3x106 cells for each of the duplicates.
Project description:<h4>Objective</h4>To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.<h4>Experimental design</h4>The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.<h4>Results</h4>PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.<h4>Conclusions and clinical relevance</h4>PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.
Project description:BACKGROUND: The aim of this current study was to assess the expression and activity of Src family kinases, focal adhesion kinase (FAK), caveolin (Cav-1) and RhoGD12 in bladder cancer. METHODS: Fifty-eight patients with a new diagnosis of bladder cancer undergoing transurethral resection were included. Immunohistochemical staining was utilised to assess expression of c-Src, dephosphorylated (SrcY(530)), phosphorylated Src (Y(419)), phosphorylated FAK (FAK Y(861)), Cav-1 and RhoGD12. Expression was assessed using the weighted histoscore method. RESULTS: High expression of dephosphorylated Y(527), phosphorylated Y(416) and phosphorylated FAK Y(861) in the membrane were associated with increased cancer-specific survival (P=0. 01, P=0.001, P=0.008, respectively) and expression of Y(416) in the membrane was an independent factor on multivariate analysis when combined with known clinical parameters (P=0.008, HR 0.288, 95% CI 0.11-0.72). CONCLUSION: These results demonstrate that in contrast to other solid tumours, activation of the Src family members and downstream signalling proteins are associated with a good prognosis in transitional cell carcinoma of the bladder, and activated Src has a positive relationship with RhoGD12.
Project description:Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Here we sequenced the exomes of nine individuals with TCC and screened all the somatically mutated genes in a prevalence set of 88 additional individuals with TCC with different tumor stages and grades. In our study, we discovered a variety of genes previously unknown to be mutated in TCC. Notably, we identified genetic aberrations of the chromatin remodeling genes (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A and CHD6) in 59% of our 97 subjects with TCC. Of these genes, we showed UTX to be altered substantially more frequently in tumors of low stages and grades, highlighting its potential role in the classification and diagnosis of bladder cancer. Our results provide an overview of the genetic basis of TCC and suggest that aberration of chromatin regulation might be a hallmark of bladder cancer.
Project description:Transitional bladder carcinoma (BCa) is prevalent in developed countries, particularly among men. Given that these tumors frequently recur or progress, the early detection and subsequent monitoring of BCa at different stages is critical. Current BCa diagnostic biomarkers are not sufficiently sensitive for substituting or complementing invasive cystoscopy. Here, we sought to identify a robust set of urine biomarkers for BCa detection. Using a high-resolution, mass spectrometry-based, quantitative proteomics approach, we measured, compared and validated protein variations in 451 voided urine samples from healthy subjects, non-bladder cancer patients and patients with non-invasive and invasive BCa. We identified five robust biomarkers: Coronin-1A, Apolipoprotein A4, Semenogelin-2, Gamma synuclein and DJ-1/PARK7. In diagnosing Ta/T1 BCa, these biomarkers achieved an AUC of 0.92 and 0.98, respectively, using ELISA and western blot data (sensitivity, 79.2% and 93.9%; specificity, 100% and 96.7%, respectively). In diagnosing T2/T3 BCa, an AUC of 0.94 and 1.0 was attained (sensitivity, 86.4% and 100%; specificity, 100%) using the same methods. Thus, our multiplex biomarker panel offers unprecedented accuracy for the diagnosis of BCa patients and provides the prospect for a non-invasive way to detect bladder cancer.
Project description:The purpose of the study was to investigate the effects of expression of a range of genes involved in apoptosis on outcome in bladder cancer. Immunohistochemistry was used to examine expression of BCL2, BAX, P53, CD40 and CD40L in archival tissues of patients included in various treatment trials for transitional cell carcinoma (TCC) of the bladder. Data were collected on 94 patients who first presented with either invasive or superficial bladder cancer. Median follow-up for alive patients was 83 months (m) (range 12-195 m). Median survival was 80 m (95% CI=56-128 m). Median survivals for the various markers were as follows: BAX-positive patients 110 m vs BAX-negative patients 18 m (P=0.0002); CD40L-positive patients 95 m vs CD40L-negative patients 45 m (P=0.04); BCL2-positive patients 44 m and BCL2-negative patients 74 m, (P=0.64); CD40-positive patients 110 m and CD40 negative patients 45 m (P=0.12); and P53 positive patients 80 m and P53 negative patients 45 m (P=0.58). In conclusion, it was seen that overexpressions of BAX and CD40L are prognostic of better survival in TCC of the bladder. Our results also raise the possibility of the future development of CD40- and CD40 ligand-based immunotherapy for bladder cancer. This study links proapoptotic and antiapoptotic markers to overall survival.
Project description:Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.
Project description:Background. The most common malignant tumor of the urinary bladder is transitional cell carcinoma (TCC). Neural precursor cell-expressed developmentally downregulated protein 9 (NEDD9) is found to be a cell adhesion mediator. P38 Mitogen-Activated Protein Kinase is a serine/threonine kinases member which can mediate carcinogenesis through intracellular signaling. Methods. To assess their prognostic role; NEDD9 and p38 protein were evaluated in sections from 50 paraffin blocks of TCC. Results. The high expressions of NEDD9 and p38 protein were significantly associated with grade, stage, distant metastasis (p < 0.001), number of tumors, lymph node metastasis, and tumor size (p < 0.001, 0.002; 0.018, <0.001; and 0.004, 0.007, respectively). High NEDD9 and p38 detection had a worse 3-year OS (p = 0.041 and <0.001, respectively). By multivariate analysis the NEDD9 and p38 protein expression levels and various clinicopathological criteria including gender, grade, stage of the tumor, and regional lymph node involvement were independent prognostic parameters of TCC of the urinary bladder patients' outcome. Conclusion. NEDD9 and p38 protein expressions were poor prognostic markers of TCC.